The health burden from beta-hemoglobinopathies and thalassemias is enormous. Increased fetal hemoglobin (HbF) ameliorates the severity of these disorders. While much progress has been made in an understanding of the "hemoglobin switch" from gamma- to beta-globin, specific intracellular regulators of this critical developmental event are unknown and the switch cannot be reliably manipulated in patients. This research is focused on new approaches to the hemoglobin switch that rely on genetic, rather than strictly biochemical or molecular, strategies. Progress in several areas suggests that the time is propitious for new initiatives. Several independent, but complementary, approaches will be taken. First, integrative genomic analysis will be applied to identify the specific locus at chromosome position Xp22 that has previously been linked to F-cell production by other investigators. Preliminary in silico analyses suggest a limited number of candidate genes within this interval. Candidates will be validated or excluded by association studies using high-density SNPs, sequencing of highly likely candidates, and functional studies in mouse erythroid cells harboring the human beta-globin locus. The aim is to identify the first trans-regulator of the hemoglobin switch. Second, high-level HbF expression is a hallmark of the rare pediatric malignancy juvenile myeloid leukemia, a disorder that arises sporadically or in the setting of Noonan's syndrome and neurofibromatosis type I. A common feature is mutation of PTPN11 or neurofibromin with consequent activation of the Ras pathway. Based on these clinical observations, the hypothesis that increased Ras activity stimulates gamma-globin production will be pursued through the study of engineered mice that have been made available for these studies. If the Ras pathway is validated as a modulator of HbF expression, this finding would open the way to consideration of new therapeutic approaches to influencing the hemoglobin switch in patients. In parallel, the potential role of the newly identified factor zfp148 in hemoglobin switching will be pursued in collaboration with Dr. Cantor (Project 4). Finally, unbiased genetic screens will be initiated to identify genes whose expression either promotes or inhibits gamma-globin expression. An appropriate "reporter" mouse erythroid cell line will be used in both genome-wide siRNA and retroviral insertional mutagenesis screens. Through these multidisciplinary approaches unrecognized regulators of the hemoglobin switch will be discovered.